Novel antifugal antibiotic substance, process for production of the same, and agricultural and horticultural fungicidal composition containing said substance

ABSTRACT

The antifungal antibiotic substance &#34;F-1028&#34; having the following formula, ##EQU1## or an acid salt thereof, a process for the production of the same by fermentation, and a fungicidal composition useful for agricultural and horticultural use.

This invention relates to a novel antifungal antibiotic substance"F-1028" and an acid salt thereof, a process for producing the same byfermentation, and a fungicidal composition useful for agricultural andhorticultural use.

We have now found that an antifungal antibiotic substance hithertounknown can be produced by cultivating "F-1028" substance-producingstrain belonging to the genus Streptomyces in a culture mediumcontaining a carbon source, a nitrogen source and a mineral underaerobic conditions.

Before this invention, the existence of a strain belonging to genusStreptomyces which can produce substance "F-1028" having strongantifungal action and growth inhibiting action on various fungi,especially molds, which cause plant diseases was not known.

The novel antifungal antibiotic substance named "F-1028" exhibits strongfungicidal activity against various fungi which cause plant diseases butonly weak antibacterial action on various bacteria.

This "F-1028" substance has the following molecular formula ##EQU2## andhas the following properties (i) - (ix); I. IT HAS A MOLECULAR WEIGHT OF219,

Ii. it is colorless crystal,

Iii. the melting point of the hydrochloric acid salt thereof is above195°C (decomposition)

Iv. the specific rotation of the hydrochloric acid salt thereof is[α]_(D) ²³ = 63.2° (c=1, H₂ O),

v. the UV spectrum of an aqueous solution of the hydrochloric acid saltthereof has no peculiar absorption as shown in FIG. 1,

vi. the IR spectrum of a mixture of the hydrochloric acid salt thereofwith potassium bromide is as shown in FIG. 2,

vii. it has an Rf value in thin-layer chromatography with silica gel of0.68 with a developing solvent consisting of n-propanol-pyridine-aceticacid-water in a ratio of 15:10:3:12; and 0.21 with a developing solventconsisting of butanol acetic acid-water in a ratio of 3:1:2,

Viii. it is readily soluble in water and insoluble in chloroform,benzene and ether, and

Ix. the color reaction is positive with ninhydrin, Ehrlich,Elson-Morgan, Tollens and Benedict reagents, and negative with Molisch,Sakaguchi, maltol and ferric chloride reagents.

This "F-1028" substance has activity against Archimycetes, Phycomycetes,Ascomycetes, Basidiomycetes, and Fungi imperfecti. It can be used forexample as a plant-protecting fungicide having both preventive andcurative effects against a wide variety of plant pathogens causing suchdiseases as blast, sheath blight, bacterial leaf blight, orhelminthosporium leaf spot of rice, leaf rust of wheat, bacterial softrot of Chinese cabbage, brown rot of peach, leaf spot of banana, graymold of strawberry and other crops, downy mildew of grape, anthracnoseof grape, apple and pear, stem rot of vegetables, anthracnose of melonsand cucumber, melanose of citrus fruits, powdery mildew of wheat, apple,and cucumber, fungi causing black spot, for example, brown spot diseaseon apple or early blight on potato, fungi causing scab, for example,scab on pear or apple.

The present inventors newly separated the "F-1028" substance-producingstrain from soil and named it Streptomyces kagawaensis.

The Streptomyces kagawaensis nov. sp. (FERM-P No. 953: ATCC No. 21811)is characterized by the following microbiological properties.

I. Morphological properties:

According to the microscopic observation, the aerial mycelium on asynthetic agar medium and a protein agar medium is branched irregularly.Sporangiopere forms closed spirals with no whorl. Spore is ellipse insize of 0.7μ × 0.9μ and has spinous surface.

II. Cultural characteristics on various culture media:

1. Sucrose-nitrate agar medium (at 27°C).

Growth; Good, pale yellowish brown to light pale yellow.

Aerial mycelium; Powdery, abundant, grayish brown.

Reverse; Yellow.

Soluble pigment; Pale brown.

2. Glucose-asparagin agar medium (at 27°C).

Growth; Good, pale yellow to pale yellowish brown.

Aerial mycelium; Powdery, abundant, pale pink to brownish white.

Reverse; Yellowish brown.

Soluble pigment; Pale yellow.

3. Glycerin-asparagin agar medium (at 27°C).

Growth; Good, yellowish brown, vegetative mycelium enters into themedium.

Aerial mycelium; White to pinkish white.

Reverse; Brown.

Soluble pigment; Yellowish brown.

4. Starch-inorganic salt agar medium (at 27°C).

Growth; Good, pale yellow.

Aerial mycelium; Powdery, brownish white to pale brown.

Reverse; Yellowish brown.

Soluble pigment; Yellowish brown.

5. Thyrosine agar medium (at 27°C).

Growth; Light yellowish brown to dark brown.

Aerial mycelium; White to brownish white.

Reverse; Brown to dark brown.

Soluble pigment; Black.

6. Nutrition agar medium (at 27°C).

Growth; Light yellowish brown.

Aerial mycelium; None.

Reverse; Brown.

Soluble pigment; Yellowish brown.

7. Yeast-malt agar medium (at 27°C).

Growth; Good, yellowish brown, vegetative mycelium enters into themedium.

Aerial mycelium; Cotton like, white to pale pink.

Reverse; Brown.

Soluble pigment; Yellowish brown.

8. Oat meal agar medium (at 27°C).

Growth; Good, yellowish brown, vegetative mycelium enters into themedium.

Aerial mycelium; Powdery, abundant, bright brown.

Reverse; Brown.

Soluble pigment; Yellowish brown.

9. Peptone-yeast iron agar medium (at 27°C).

Growth; Lustrous yellowish white.

Aerial mycelium; None.

Reverse; Grayish brown.

Soluble pigment; Dark brown.

10. Loeffler coagulated serum agar medium (at 27°C).

Growth; Lustrous brown.

Aerial mycelium; Scant, white.

Soluble pigment; Brown, no liquefaction.

11. Egg agar medium (at 27°C).

Growth; Creased, yellowish brown to dark brown.

Aerial mycelium; White to dim white.

Reverse; Brownish brown.

Soluble pigment; Dark yellowish brown, no liquefaction.

12. Potato plug (at 27°C).

Growth; Thick, raised lustrous yellowish brown.

Aerial mycelium; Scant, white.

Soluble pigment; Yellowish brown to dark brown.

13. Carrot plug.

Growth; Thick, raised, lustrous yellowish brown.

Aerial mycelium; Cotton like, white.

Soluble pigment; Scant, pale yellowish brown.

14. Glucose-peptone gelatin medium (at 27°C).

Growth; Creased, pale grayish brown.

Aerial mycelium; Pale grayish white.

Soluble pigment; Brown.

15. Skim milk medium (at 27°C).

Growth; Ring or membrane, pale yellowish brown.

Aerial mycelium; Scant, white.

Soluble pigment; Pale yellowish brown.

III. Physiological properties:

1. Optimum conditions for growth:

Temperature: between 25° to 30°C.

pH: between 6 to 8.

Aerobic condition.

2. Growable conditions:

Temperature: between 15° to 38°C.

pH: between 4 to 9.

3. Formation of melanine: positive.

4. Reduction of nitric acid: weak.

5. Milk coagulation: weak.

6. Milk-peptonization: moderate.

7. Gelatin-liquefaction: weak.

8. Decomposition of starch: positive.

9. Formation of hydrogen sulfide: positive.

10. Dissolution of coagulated serum: negative.

IV. Assimilation of carbon sources:

The assimilation of carbon sources was tested by the method of D. G.Pridham et al.

Good assimilation:

Arabinose, glucose, galactose, glycerol, levulose, mannose, maltose,melibiose, sucrose, trehalose, inositol, mannitol.

Moderate assimilation:

Lactose, xylose.

Slight assimilation:

Raffinose, rhamnose, salicin, inulin, sorbinol.

No assimilation:

Melezitose, sorbose, adonitol, dulcitol.

According to these microbiological properties, the "F-1028"substance-producing strain, which grows with pale pink or grayish brownaerial mycelium and produces a brown or yellowish brown soluble pigmenton a protein agar medium, is considered to below to Strepromyces,particularly Streptomyces lavendulae.

Among known species of Streptomyces having these characteristicproperties, Streptomyces lavendulae, Streptomyces venezuelae andStreptomyces verginiae, described in Bergey's Mannual of DeterminativeBacteriology, 7th ed. and the Actimomycetes by Waksman, Vol. 2, aresimilar to the strain of this invention.

However, the spore surfaces of these three strains are smooth, but onthe contrary, that of the "F-1028" substance-producing strain isspinous. Comparison of the cultural properties between the "F-1028"substance-producing strain and these three strains shows that the threestrains produce no soluble pigment but the "F-1028" substance-producingstrain produces yellowish brown soluble pigment on sucrose-nitrate agarmedium. Also, in the assimilation of carbon sources, any of these threestrains shows little or no assimilation of merbiose, sucrose, inositoland mannitol, but the "F-1028 substance-producing strain assimilatesthese saccharides well.

By the differences of the morphological, cultural and physiologicalproperties, the "F-1028" substance-producing strain can be clearlydistinguished from Streptomyces lavendulae, Streptomyces venezuelae andStreptomyces verginia. The present inventors classified the "F-1028"substance-producing strain as Streptomyces lavendulae group in view ofthe cultural and physiological properties, but distinguished it as a newstrain from the known microorganisms under the name of Streptomyceskagawaensis nov. sp., according to the differences of spore surfacestructure and saccharide-assimilation and the possibility of producing anew antibiotic "F-1028" substance.

Streptomyces kagawaensis is easily converted to a mutant strain by meansof artificial mutations such as application of an ultraviolet ray,X-rays, or a high frequency wave, or radial rays, chemicals.

In the process of the present invention, there are employed all strains,which have a productivity of "F-1028" substance, including mutants of astandard strain which are not clearly distinguished from Streptomyceskagawaensis as a standard strain.

The important aspect of the process of the present invention is based onthe steps of culturing the "F-1028" substance-producing strain belongingto Streptomyces kagawaensis and isolating the antibiotic "F-1028"substance from the culture medium.

In this invention, the "F-1028" substance-producing strain is culturedin a culture medium containing nutrients utilized by generalmicroorganisms. As the nutrient source, all of the known nutrients foractinonyces can be used, for example, commercially available glucose,starch, glycerol, sucrose, maltose, dextrin, molasses and fats as carbonsources (shown in Table 1).

Examples of the nitrogen source are commercially available soybeanflour, meat extract, peptone, yeast, corn steep liquor, powdered cottonseed, powdered peanut, N-Z amine, casein, ammonium sulfate, ammoniumnitrate and sodium nitrate (shown in Table 2).

Examples of the minerals (or inorganic salts) are calcium carbonate,sodium chloride, potassium chloride, phosphate, magnesium sulfate. Ifdesired, a trace amount of metallic salts can be incorporated.

Any substance of these nutrients may be used, if it is assimilated bythe "F-1028" substance-producing strain and useful for producing the"F-1028"substance.

Production of the "F-1028" substance in a culture medium containingvarious carbon sources will now be described.

In a test tube (20 mm in diameter) was placed 13 ml of a liquid culturemedium containing a basic medium, which consisted of peptone 0.5 %, meatextract 0.5 %, dried yeast 0.3 %, sodium chloride 0.5 % and calciumcarbonate 0.3 %, and the carbon sources described in the following table(shown in Table 1). Then the seed culture of the "F-1028"substance-producing strain was inoculated to the culture medium. Theinoculated test tube was incubated with shaking in a reciprocated shakerat 27°C. The production of the "F-1028" substance is shown in Table 1.

                  Table 1                                                         ______________________________________                                                    Two days   Three days                                                                   F-1028         F-1028                                   Carbon source pH      mcg/ml   pH    mcg/ml                                   ______________________________________                                        1   Glucose  2.0 %    7.4   632    7.6   280                                  2   Glycerol 2.0 %    7.4   452    7.4   258                                  3   Starch   2.0 %    7.6   548    8.0   148                                  4   Glucose  1.0 %    7.4   538    8.0   184                                      Starch   1.0 %                                                            5   Glycerol 1.0 %    7.6   590    8.0   210                                      Starch   1.0 %                                                            6   Maltose  2.0 %    8.2    78    8.4    32                                  7   Dextrin  2.0 %    7.8   284    7.8   420                                  8   Sucrose  2.0 %    8.4    36    8.4                                        ______________________________________                                    

Production of the "F-1028" substance in a culture medium containingvarious nitrogen sources will now be described.

To a basic medium consisting of glucose 2.0 %, sodium chloride 0.5 %,calcium carbonate 0.3 %, the nitrogen sources as shown in the followingtable were added. The culture medium was adjusted to pH 7, sterilized,inoculated with the seed culture of the "F-1028" substance-producingstrain and shaken at 27°C. The production of the "F-1028" substance isdescribed in Table 2.

                  Table 2                                                         ______________________________________                                                     Two days   Three days                                                                   F-1028         F-1028                                  Nitrogen source                                                                              pH      mcg/ml   pH    mcg/ml                                  ______________________________________                                        1   Peptone    1.0 %   7.6   632    7.8   420                                 2   Meat extract                                                                             1.0 %   7.8   1212   7.8   358                                 3   Powdered                                                                      soybean    3.0 %   7.4   580    7.8   322                                 4   Corn steep                                                                    liquor     2.0 %   7.4   368    7.8   174                                 5   Dried yeast                                                                              1.0 %   6.8   --     6.4   116                                 6   Peptone    0.5 %                                                              Meat-extract                                                                             0.5 %   7.6   322    7.8   342                                     Dried yeast                                                               ______________________________________                                    

The best cultural method uses a liquid culture, expecially a submergedtank culture with aeration at temperatures ranging over the extent inwhich the "F-1028" substance-producing strain is growable and producesthe "F-1028" substance, preferably at 25° to 35°C. The practical cultureis conducted at about 27°C and continued until the "F-1028" substance issufficiently accumulated. The pH value of the cultural medium is usuallyfrom 6 to 9, preferably 6.2 to 8.5, especially 6.5 to 8. Theconcentration of the "F-1028" substance in the culture medium becomesmaximum within 2 to 5 days by both shaking culture and tank culturemethod. However, since the days needed to get the maximum concentrationof the "F-1028" substance may be varied in accordance with condition ofaeration and agitation in the same culture medium at the sametemperature, it is desirable to determine the potency of the "F-1028"substance. When the potency is maximum, the culture is stopped and the"F-1028" substance is extracted. To determine the potency of the"F-1028" substance, there was employed a method of measuring aninhibition circle by using Sclerotinia cinerea (hereinafter referred toas Sc) in accordance with a conventional way.

The practice was conducted as follows:

One Kg of potato and 15 lit. of tap water were boiled for 30 minutes andthe potatos were filtered away by gauze. Then to the filtrate, water wasadded in an amount of 5 lit. and commercially available sucrose wasincorporated at 2 % to prepare a culture medium for Sc. To this liquidmedium agar was added at 1.5 % and it was placed in a test tube in orderto make a slant culture medium. Sclerotinia cinerea strain wasinoculated on the slant medium and incubated for 4 to 7 days at 27°C and2 or 3 loopful of Sc strain on the slant culture medium were inoculatedin 100 ml of Sc culture medium containing agar at 0.3 % in a Sakaguchi'sflask. After the flask was shaken for 4 days at 27°C, the cultivatedmedium was homogenized for 2 minutes at 9,000 r.p.m. It was used thenfor seeding. Thereafter, 10 ml of the Sc liquid medium added agar at 1to 1.2 % was placed in a petri dish, solidified and covered with 4 ml ofthe same agar medium mixed with the cultivated medium at 10 %. Thisplate culture medium was used in a conventional disc method. The culturewas continued for 2 days at 27°C. In this method, a 200 mcg/ml solutionof the pure "F-1028" substance forms an inhibition zone with about 35 mmdiameter. A linear relationship between the concentration of theantibiotic and the diameters of inhibition zones stands at a range 10 to200 mcg/ml.

Since the "F-1028" substance has physical and chemical properties asdescribed hereinafter, the substance can be extracted and purified inaccordance with the properties. The "F-1028" substance was produced inthe culture liquid, the mycelia are removed away by methods ofcentrifuging or filtration with auxiliary filters such as kieselguhr atacidity or neutrality. Then the "F-1028" substance was adsorbed on acation exchange resin, for example IRC-50(Na⁺) or IR-120(H⁺), by thebatch or column method, and eluted with acidic or basic water. The"F-1028" substance is a strong basic compound which is not substantiallyadsorbed on IRC-50(H⁺), but it is adsorbed on the resin in Na⁺ type. Forinstance, the filtrate of the fermentation mixture is treated withoxalic acid, and adsorbed on IRC-50(Na⁺), and the antibiotic was elutedrapidly with 1 N-HCL. In the case of using activated carbon, the"F-1028" substance can be adsorbed on the carbon and eluted with aqueousacetone and aqueous methanol but not substantially.

After the eluate containing the "F-1028" substance was concentratedunder reduced pressure, the concentrate, if necessary, was desalted withmethanol, ethanol and added with an exessive amount of methanol ethanoland acetone to precipitate the "F-1028" substance in the form of powder.The eluate from the resin decomposed easily with coloring in brown bydrying directly under reduced pressure. It was necessary to treat the"F-1028" substance rapidly, because its activity became lower and itcoloured in yellow when it was allowed to stand for a long time. Themixture containing the "F-1028" substance was decomposed in brown byprecipitation with methanol, ethanol and acetone and drying. However,the "F-1028" substance is very stable as far as it is in the form ofaqueous solution. The crude powder obtained was purified by methods suchas carbon chromatography or Sephadex chromatography to obtain a whitepowder.

The eluate as hydrochloride was then passed through a column of IR-4Bresin to be converted to a free type and thereafter to a sulfate byeluting with sulfuric acid. In this case, if the amount of IR-4B resinwas not sufficient and the eluates from IR-4B resin were passed througha column of Sephadex G-10, two fractions were eluted from the Sephadexcolumn. The first fraction was a sulfate of the "F-1028" substance andthe second was a hydrochloride. The latter fraction was again passedthrough the column of IR-4B resin and eluted with sulfuric acid and theeluate was subjected to the Sephadex column to form a sulfate. The"F-1028" substance isolated and purified by the methods described above,has the following more detailed physical and chemical properties.

1. Colorless needle-like or plate-like crystal.

2. Melting point (hydrochloride), decomposed at above 195°C.

3. elemental analysis (as hydrochloride):

Found: C: 30.94%, H: 6.58%, N: 13.39%, 0: 25.91%, Cl: 23.18%.

4. Molecular formula: C₈ H₁₇ N₃ 0₄. (molecular weight: 219, fromelemental analysis, titration and NMR spectrum).

5. Equivalent: 122.5 (from titration).

6. Specific rotation (hydrochloride):

[α]_(D) ²³ = 63.2° (C=1, H₂ 0).

7. pka': 7.02 and 8.16.

8. Ultraviolet absorption spectrum:

No peculiar absorption as shown in FIG. 1.

9. infrared absorption spectrum:

As shown in FIG. 2.

10. rf value:

When a thin layer chromatography based on silica gel is used with adeveloping solvent consisting of butanol, acetic acid and water in aratio 3:1:2, Rf value is 0.25. When the solvent consists of n-propanol,pyridine, acetic acid and water in a ratio of 15:10:3:12, Rf value is0.68.

11. Solubility:

Readily soluble in water;

Soluble in methanol and dimethyl sulfoxide;

sparingly soluble in ethanol;

Insoluble in chloroform, benzene and ether.

12. Color reaction:

Positive: Ninhydrin, Ehrlich, Elson-Morgan, Tollens, and Benedictreactions.

Negative: Molisch, Sakaguchi, maltol, and ferric chloride reactions.

13. It retains its activity in an acidic solution, but the activity isreduced when the solution becomes neutral to alkaline.

Biological properties of the "F-1028" substance

1. Antimicrobial activity:

The minimum inhibitory concentration (MIC) of the "F-1028" substance forvarious microorganisms was examined by the agar dilution method, and theresults are given in Table 3.

                  Table 3                                                         ______________________________________                                                           24 hours   48 hours                                        Microorganism      mcg/ml     mcg/ml                                          ______________________________________                                        Staphylococcus aureus FDA 209 p                                                                  >100       >100                                            Sarcina lutea PCI 1001                                                                           3.12       3.12                                            Bacillus subtilis PCI 219                                                                        >100       >100                                            Mycobacterium ATCC 607                                                                           >100       >100                                            Escherichia coli NIHJ                                                                            100        >100                                            Vibrio comma 904   12.5       100                                                                48 hours   72 hours                                        ______________________________________                                        Candida albicans   >100       >100                                            Saccharomyces cerevisiae                                                                         >100       >100                                            Holmodendrum pedrosei                                                                            25         25                                              Trichophyton rubrum                                                                              100        >100                                            Ophiobolus miyabianus                                                                            25         25                                              Aspergillus niger  >100       >100                                            Xanthomonas oryzae 50         100                                             Xanthomonas citri  >100       >100                                            Alternaria japonica                                                                              50         50                                              Alternaria kikuchiana                                                                            >100       >100                                            Sclerotinia cinerea                                                                              12.5       25                                              Sclerotinia sclerotiorum                                                                         1.56       3.12                                            Botorytis fabae    6.25       25                                              Botorytis cinerea  6.25       12.5                                            ______________________________________                                    

Toxicity

i. No abnormality.

Mice (intravenous injection) 125 mg/kg, (oral administration) 500 mg/kg.

Young carp: no trouble with 250 ppm

ii. Mice (intravenous injection) LD₅₀, 160 mg/kg, (oral administration)LD₅₀, 750 mg/kg.

Accordingly, the "F-1028" substance has very low toxicity. Since thesephysiological, chemical and biological properties of the "F-1028"substance do not correspond with those of the known numerousantibiotics, the antibiotic, "F-1028" substance is confirmed to be a newantibiotic substance.

The "F-1028" substance of the invention can be applied effectively topathogens living on the above-ground portions of plants, pathogens whichinvade plants from the soil and cause tracheomycosis, seed-infectiouspathogens, and soil-infectious pathogens.

As mentioned before, the "F-1028" substance of this invention exhibitssuperior control effect against pathogens which invade rice plants andother crops, and has good affinity for higher plants. With ordinarydosages, it does not give phytotoxicity to crops, and hardly exhibitstoxicity on domestic animals and fish. Thus, this substance can be usedagainst plant pathogens as agricultural and horticultural fungicidequite conveniently, and is practically very valuable for increasingagricultural productivity.

In the application of the "F-1028" substance of this invention as anagricultural and horticultural fungicide, it can be used after dilutionwith water, or as various formulations prepared by the method usuallypractised in the field of producing agricultural chemicals usingagricultural chemical assistants. These various formulations may beapplied as such or after dilution with water to the desiredconcentrations.

As the agricultural and horiticultural assistants referred to herein,there can be named inert solvents and/or diluents (extenders orcarriers) (these are used to carry the active ingredient to pathogenicfungi and/or the habitat of the pathogenic fungi). There can also becited various surface active agents and/or organic materials, such asstabilizers, spreaders (stickers), propellants for aerosols, orsynergizing agents (these are used to produce, maintain, and promote theeffect of the active compound to a greater extent).

Examples of the solvent include water, and organic solvents, forexample, aliphatic or alicyclic hydrocarbons such as n-hexane,industrial gasolines (petroleum ether, solvent naphtha, etc.), petroleumfractions (paraffin wax, kerosene, light oil, middle oil, heavy oil,etc.), aromatic hydrocarbons such as benzene, toluene, xylenes, oraromatic naphtha, halogenated hydrocarbons such as chloromethylene,chloroethylene, carbon tetrachloride, trichloroethylene, ethylenedibromide, or chlorobenzene, alcohols such as methyl alcohol, ethylalcohol, propyl alcohol, or ethylene glycol, ethers such as ethyl ether,ethylene oxide, or dioxane, alcohol ethers such as ethylene glycol ormonomethyl ether, ketones such as acetone and isophorone, esters such asethyl acetate or amyl acetate, amides such as dimethyl formamide ordimethyl acetamide, and sulfoxides such as dimethyl sulfoxide.

Examples of the diluents (extenders or carriers) include vegetablepowders, mineral powders, clay minerals such as kaolinites,montmorillonites or attapulgites, talc, pyrophyllite, mica, gypsum,calcite, muscovite, vermiculite, dolomite, apatite, slaked lime,magnesium lime, diatomaceous earth, inorganic acid salts such as calciumcarbonate, sulfur, pumice, synthetic mineral powders such as highlydispersed silicic acid or synthetic alumina, synthetic resins such asphenolic resins, urea resins, or vinyl chloride resins.

Examples of the surface active agents include anionic surface activeagents such as alkylsulfuric acid esters (for example, sodiumlaurylsulfate), arylsulfonic acids (for example, alkylarylsulfonic acidsalts or sodium alkylnaphthalenesulfonate), cationic surface activeagents such as alkylamines (for example, laurylamine, stearyl trimethylammonium chloride, or alkyl dimethylbenzylammonium chlorides), orpolyoxyethylene alkyl amines, non-ionic surface active agents such aspolyoxyethylene glycol ethers (for example, polyoxyethylene alkylarylethers, or polyoxyethylene alkylphenyl ethers), polyhydric alcoholesters (for example, polyoxyethylene sorbitan monolaurate), orpolyoxyethylene glycol esters (for example, polyoxyethylene fatty acidesters), and amphoteric surface active agents.

As the examples of the organic materials, there can be citedstabilizers, spreaders (stickers) such as agricultural soap, coumaroneor indene resins, or polyvinyl butyl ether, propellants for aerosolssuch as halogenated hydrocarbons (Freon, etc.), combustion agents forfumigants, such as nitrous acid salts, zinc powder, or dicyandiamide,oxygen-yielding agents such as perchlorates or bichromates,phytotoxicity-reducing agents such as zinc sulfate, ferrous chloride, orcopper nitrate, effect-extending agents such as chlorinated terphenyl,dispersion stabilizers such as casein, tragacanth, carboxymethylcellulose, or polyvinyl alcohol, and synergizing agents.

The compound of this invention can be prepared into various formulationsby the method generally practised in the field of producing agriculturalchemicals.

Examples of the formulations are liquid preparations, such asemulsifiable concentrates, wettable powders, tablets, soluble powders,or solutions, dusts, granules, fumigants, or aerosols.

The fungicide of this invention may contain the above active ingredientin an amount of 0.1 to 95% by weight, preferably 0.5 to 90% by weight.

In actual application, the suitable amount of the active ingredientcontained in the above-mentioned various formulations or in aready-to-use preparation is generally from 0.0001 to 20% by weight,preferably from 0.005 to 10% by weight.

The content of the active ingredient may be changed properly accordingto the formulations, the method, purpose, time, and place ofapplication, and the state of disease occurrence, etc.

If desired, the compound of this invention may further contain otheragricultural chemicals such as insecticides, fungicides, acaricides,nematocides, antivirus agents, herbicides, plant growth modifiers, orattractants (for example, organophosphorus esters, carbamates, dithio(or thiol) carbamates, organic chlorides, dinitro compounds, organicsulfur or metal compounds, antibiotics, substituted diphenyl ethercompounds, urea compounds, or triazine compounds), and/or fertilizers.

Various formulations or ready-to-use preparations containing the activeingredient of this invention described above can be applied by themethod generally practised in the field of agricultural chemicals, suchas spraying (for example, liquid preparation spraying, misting,atomizing, dust spraying, granule spraying, application to the watersurface, or pouring), fumigating, application to the soil (for example,sprinkling, vaporing, or pouring), application to the surface of thesoil (for example, coating, banding, dust application, or covering), orimpregnation. They can also be applied by the ultra-low-volume sprayingmethod. According to this method, the active ingredient can be includedin an amount of up to 95% or even 100%.

The amount of application per unit area of the fungicide of thisinvention is about 3-1000 g, preferably 30-600 g, per 10 areascalculated as the active compound. But in special cases, the amount mayeither be above or below this range, or sometimes, it is even necessaryto deviate from this range.

According to this invention, there is provided a fungicidal compositioncomprising the compound of formula (I) as an active ingredient, and asolvent and/or a diluent (extender or carrier) and/or a surface activeagent, and if desired, an organic material.

Furthermore, the present invention provides a method of controllingpathogenic fungi, which comprises applying the compound of formula (I)singly or in admixture with a solvent and/or a diluent (extender orcarrier) and/or a surface active agent, and if desired, further with anorganic material to said fungi and/or their habitat.

The present invention will be specifically described by the followingExamples, but it should be understood that the invention is not limitedin any way to these Examples.

FIG. 1 shows a UV spectrum curve of an aqueous solution of thehydrochloride of the antibiotic, the "F-1028" substance, of thisinvention.

FIG. 2 shows a IR spectrum curve of the hydrochloride of the "F-1028"substance mixed with potassium bromide.

EXAMPLE 1

A loopful micelium of Streptomyces kagawaensis, the "F-1028"substance-producing strain, was inoculated to a cultural mediumcontaining meat extract 0.5 %, sodium chloride 0.5 %, yeast extract 0.3%, glucose 2 %, peptone 0.5 % and calcium carbonate. After theinoculated medium was cultivated with shaking for 2 days, it was placedin a 100 lit.-fermentation tank with 75 lit. of the same culture medium.Then this inoculated medium was incubated at 27°C with aeration of 10lit./min, agitation of 250 r.p.m., and internal pressure of 0.5 kg/cm²for 2 days. The production of the "F-1028" substance then reached 400mcg/ml. in the broth. 70 lit. of the cultivated medium was adjusted topH 2 by a saturated solution of oxalic acid and filtered with celite at0.5 %. Then the filtrate was adjusted to pH 5 with 2N-NaOH and stirredtogether with 2.8 lit. of cation exchange resin, Amberlite IRC-50(Na⁺),for 30 minutes. Successively, the resins were collected by filtration,and the filtrate was adjusted again to pH 5, admixed with 1.4 lit. ofAmberlite IRC-50(Na⁺), stirred and filtered. These resins werecollected, too. By two of these operations. 95 % of the "F-1028"substance in the filtrate was adsorbed by the resin. Thereafter, theresin which adsorbed the "F-1028" substance was washed sufficiently withwater and charged into a column. The "F-1028" substance was eluted with1N-HCL, and about 5 lit. of the eluate containing the active substancewas collected and concentrated to 100 ml under reduced pressure. Afterthe filtration of sodium chloride formed in this step, three-foldquantity of methanol was added to the solution. It was allowed to standunder cold conditions. The sodium chloride further formed was filteredaway, ten-fold quantity of acetone was added. Then the precipitate ofthe "F-1028" substance was formed and 95 g of the "F-1028" substance ascrude powder (Yield: 60 to 70 %) was obtained by drying the precipitate.

EXAMPLE 2

125 ml of a medium containing glucose 2 %, soy-bean meal 3 %, sodiumchloride 0.5 % and calcium carbonate 0.5 % were placed in a 500ml-Sakaguchi's flask sterilized and inoculated by 2 ml of precultivatedmedium of Streptomyces kagawaensis, the "F-1028" substance-producingstrain. Then the flasks were incubated at 27°C with shaking at 120r.p.m. and with 8 cm amplitude. After 48 hours, potency of the "F-1028"substance became 580 mcg/ml. The broth was collected and the activecompound was extracted by the same methods as in Example 1 to be a crudepowder.

EXAMPLE 3

A mutant of Streptomyces kagawaensis, obtained by treatment withN-methyl-N'-nitro-N-nitrosoguanidine and grew with pink or brownish grayaerial mycelia, was precultured. And the cultivated medium wasinoculated by 2 ml in 500 ml-Sakaguchi's flask placed with 125 ml ofcultural medium containing glucose 2 %, peptone 0.5 %, meat extract 0.5%, dried yeast 0.3 %, sodium chloride 0.5 %, calcium carbonate 0.3 %.Then the flasks were incubated at 27°C with shaking at 120 r.p.m. with 8cm amplitude. By the same procedure as in Example 1, the active compoundwas extracted to obtain the "F-1028" substance in a crude powder. Theresults were shown in Table 4.

                  Table 4                                                         ______________________________________                                                     2 days     3 days                                                                       F-1028         F-1028                                  Strain         pH      (mcg/ml) pH    (mcg/ml)                                ______________________________________                                        Monospore-culturing                                                           pinky brown M 1 strain                                                                       8.0     1580     8.0    736                                    Monospore-culturing                                                           pale pink M 2 strain                                                                         8.4     2160     8.2   2220                                    Nitrosoguanidine-                                                             treated brownish                                                              gray N 1 strain                                                                              7.2     1106     7.8    474                                    Nitrosoguanidine-                                                             treated white N 2                                                             strain         6.2      452     7.6   2000                                    ______________________________________                                    

EXAMPLE 4

6 g of the crude powdered "F-1028" substance, obtained in Example 1 wasdissolved in 10 ml of water, adsorbed on a column filled with 60 g ofactivated carbon for chromatography (produced by Wako Seiyaku Co., Ltd.)and developed with water, and the "F-1028" substance was eluted in acolorless aqueous solution (Yield: 91.5 %). Then the solution wasconcentrated in vacuo and admixed with about 20-fold quantity ofethanol. The precipitate was dried to obtain 4 g of the crude "F-1028"substance in powder. Successively, 2 g of the powder was dissolved in 4ml of water, poured into Sephadex G-10 column (2.5 × 150 cm) anddeveloped with water. The active fraction was concentrated in vacuo toobtain 1.15 g of white powder. A methanol solution of the white powderwas allowed to stand to precipitate crystals. 170 mg of plate-like orneedle-like crystals of the "F-1028" substance were obtained ashydrochloride.

EXAMPLE 5

An aqueous solution of the hydrochloride of the "F-1028" substance,eluted from the activated carbon column by the procedure as described inExample 4, was passed through a resin column filled with IR-4B[OH^(-]).The solution passed through the column was neutralized with sulfuricacid, concentrated and admixed with ten-fold quantity of ethanol oracetone. There was obtained a sulfate of the "F-1028" substance inpowder. The powder was then applied to Sephadex G-10 column to beseparated into two fractions. The former was the sulfate, and the latterwas the hydrochloride. The latter was again passed through IR-4B[OH^(-])column. The solution passed through the column was neutralized withsulfuric acid to obtain the sulfate. An aqueous solution of the sulfatewas concentrated to dryness in vacuo to form a white powder.

The excellent features and marked effects of the active compound of thisinvention, as an agricultural and horticultural fungicide, can berecognized from the results of the following tests in which it was usedagainst various pathogenic fungi.

EXAMPLE 6

Test on sclerotinia rot of beans and gray mold on vegtables (preventivetest):

The hydrochloride of "F-1028" substance of a prescribed concentrationwas sprayed to bean (variety: Taisho Kintoki) in the 2-3 leaf stage at arate of 15 cc per pot. One day after the spraying, the leaves were cutoff, and the hypha growing portion of gray mold fungus and sclerotiniarot fungus which had previously been cultivated in a PDA plate culturemedium was punched out in a size of 7 mm in diameter and 2 mm inthickness, and inoculated thereto. The inoculated leaves were placed ina constant-temperature vessel (at 20°C., humidity of more than 98%), andafter a lapse of 3 days, the diameters of the enlarged disease spotswere measured. Then, the disease spot inhibiting rate was calculated.The results are shown in Table 5 below.

The disease spot inhibiting rate was calculated as follows: ##EQU3##

                                      Table 5                                     __________________________________________________________________________                                 Sclerotinia                                                        Gray mold fungus                                                                         rot fungus                                                  Concent-                                                                             Diameter                                                                            Inhibi-                                                                            Diameter                                                                            Inhibi-                                               ratio of                                                                             of disease                                                                          ting of disease                                                                          ting                                                  active spot  rate spot  rate                                       Compound   ingredient                                                                           (mm)  (%)  (mm)  (%)                                        __________________________________________________________________________    F-1028 substance,                                                                        50 ppm 10    67   0     100                                        hydrochloride                                                                            25 ppm 23    23   10    71                                         F-1028 substance,                                                                        Original                                                                             11    63   0     100                                        culture liquor                                                                           liquid                                                                        8 X    18    40   0     100                                        Allisan (50%)*                                                                           × 1000                                                       (commercially                                                                 available, control)                                                                      (500 ppm)                                                                            10    67   3     91                                         Polyoxyne (5%)                                                                           × 1000                                                       (commercially                                                                 available, control)                                                                      (50 ppm)                                                                             19    37   20    57                                         Non-treated       30     0   35    0                                          __________________________________________________________________________     *Allisan : 2,6-dichloro-4-nitroaniline.                                  

EXAMPLE 7

Test on gray mold and sclerotinia rot of beans (curative test):

Leaves of bean (variety: Taisho Kintoki) were cut off, and gray moldfungus and sclerotinia rot fungus which had been cultivated in a PDAplate culture medium, punched out in a size of 7 mm in diameter and 2 mmin thickness, were inoculated thereto. The inoculated leaves were placedfor 24 hours in a humid chamber at 20°C. The diameters of the resultantdisease spots were measured.

"F-1028" substance diluted to a prescribed concentration was sprayed tothe leaves to an extent such that it trickled from the leaves. Theleaves were allowed to dry in air, and again were placed in a constanttemperature vessel at 20°C. After a lapse of 48 hours, the diameters ofthe disease spots were calculated. The degree of progress of the diseasespots as against the disease spots before spraying of the chemical wasexpressed by the disease spot inhibiting rate as follows: ##EQU4## dc₁ :diameter of the disease spot of non-treated leaf before application ofthe chemical

dc₂ : diameter of the disease spot of non-treated leaf after applicationof the chemical

dt₁ : diameter of the disease spot before application of the chemical

dt₂ : diameter of the disease spot after application of the chemical

The results obtained are shown in Table 6.

                                      Table 6                                     __________________________________________________________________________    (A) Test results regarding gray mold                                                             Diameter of                                                                          Diameter of                                                            the disease                                                                          the disease                                                    Concentration                                                                         spot before                                                                          spot after                                                                           Disease spot                                            of active                                                                             spraying                                                                             spraying                                                                             inhibiting                                   Compound   ingredient                                                                            (mm)   (mm)   rate (%)                                     __________________________________________________________________________    F-1028 substance                                                                         100 ppm 14.0   16.3   81                                                       50     14.0   17.0   75                                                       25     14.0   19.3   59                                           Allisan (50%)                                                                            × 1000                                                                          12.6   18.0   43                                           (commercially                                                                            (500 ppm)                                                          available, control)                                                           *Sclex (20%)                                                                             × 1000                                                                          14.0   17.7   61                                           (commercially                                                                            (200 ppm)                                                          available, control)                                                           Non-treated        11.0   23.3    0                                           __________________________________________________________________________    (B) Test results regarding sclerotinia rot                                                       Diameter of                                                                          Diameter of                                                            the disease                                                                          the disease                                                    Concentration                                                                         spot before                                                                          spot after                                                                           Disease spot                                            of active                                                                             spraying                                                                             spraying                                                                             inhibiting                                   Compound   ingredient                                                                            (mm)   (mm)   rate (%)                                     __________________________________________________________________________    F-1028 substance                                                                         100 ppm 11.3   16.3   76                                                       50     11.3   19.6   62                                           Allisan (50%)                                                                            × 1000                                                                          13.6   26.6   62                                           (commercially                                                                            (500 ppm)                                                          available, control)                                                           Non-treated        12.0   33      0                                           __________________________________________________________________________     *Sclex : 3-(3,5-dichlorophenyl)-5,5-dimethyloxazolidinedione-2,4.             EXAMPLE 8 Test on Brown rot (Sclerotinia cineria) of peach (field test):

An aqueous solution of the "F-1028" substance with a fixed concentrationwas sprayed twice with a 7-day interval to peach trees (variety:Kurakata early-ripening, 5th year stage). Both the morbidity uponharvest and morbidity of the fruit packed in a cardbord box for storageat room temperature were investigated. The results are shown in thefollowing table 7.

                                      Table 7                                     __________________________________________________________________________    Test results against Brown rot                                                                           Morbidity after                                                Spraying                                                                              Morbidity                                                                            7 days since                                       Sample compound                                                                           concentration                                                                         upon harvest                                                                         packaging in                                       __________________________________________________________________________    F-1028 substance                                                                          50 ppm  0      0                                                  BENLATE (50%)                                                                             × 1000                                                                          0.3    46.1                                               (commercial, control)                                                         SCLEX (20%) × 1500                                                                          0.03   7.7                                                (commercial, control)                                                         Untreated plot                                                                            --      38.5   61.6                                               __________________________________________________________________________     Remarks:                                                                      BENLATE: methyl-1-(butyl carbamoyl)-2-benzimidazole carbamate.                SCLEX: 3-(3,5-dichlorophenyl)-5,5-dimethyloxazolidine dione-2,4.         

We claim:
 1. The antifungal antibiotic substance "F-1028" having thefollowing formula, ##EQU5## or an hydrochloric acid salt or sulfuricacid salt thereof, which has the following properties (i) -(ix):i.molecular weight of 219, ii. colorless crystal iii. melting point of thehydrochloric acid salt thereof is above 195°C (decomposition), iv.specific rotation of the hydrochloric acid salt thereof is [α]_(D) 23 =63.2° (c=1, H₂ O), v. UV spectrum of an aqueous solution of thehydrochloric acid salt thereof has no peculiar absorption as shown inFIG. 1, vi. IR spectrum of a mixture of the hydrochloric acid saltthereof with potassium bromide is as shown in FIG. 2, vii. Rf value inthin-layer chromatography with silica gel is 0.68 with a developingsolvent consisting of n-propanol-pyridine-acetic acid-water in a ratioof 15:10:3:12; and 0.21 with a developing solvent consisting ofbutanol-acetic acid-water in a ratio of 3:1:2, viii. readily soluble inwater and insoluble in chloroform, benzene and ether, and ix. colorreaction is positive with ninhydrin, Ehrlich, Elson-Morgan, Tollens andBenedict reagents, and negative with Molisch, Sakaguchi, maltol andferric chloride reagents.
 2. The antifungal antibiotic substance ofclaim 1 wherein the acid salt is the hydrochloric acid salt.